In Vivo Analysis of Zebrafish Innate Immunity

  • Jean-Pierre Levraud
  • Emma Colucci-Guyon
  • Michael J. Redd
  • Georges Lutfalla
  • Philippe Herbomel
Part of the Methods in Molecular Biology™ book series (MIMB, volume 415)

Summary

Among vertebrate model species, the zebrafish embryo combines at an unprecedented level optical accessibility with easy genetic manipulation. As such, it is gaining recognition as a powerful model to study innate immunity. In this chapter, we provide a protocol for the generation of zebrafish embryos deficient in a protein of interest for innate immune signaling using antisense morpholino oligonucleotides, the systemic or local infection of these embryos with bacteria, and the assessment of various aspects of the following immune response with emphasis on microscopic observation. This example can be easily adapted to study the role of other genes, either knocked down or overexpressed, and in response to any other challenge, from purified microbial compounds to pathogenic viruses. This protocol is aimed at people not necessarily familiar with zebrafish biology and handling.

Key Words

Zebrafish innate immunity macrophage granulocyte morpholino experimental infection chemotaxis DIC microscopy 

References

  1. 1.
    Traver, D., Herbomel, P., Patton, E., Murphey, R., Yoder, J., Litman, G., Catic, A., Amemiya, C., Zon, L., and Trede, N. (2003) The zebrafish as a model organism to study development of the immune system. Adv. Immunol. 81, 253–330.PubMedGoogle Scholar
  2. 2.
    Trede, N., Langenau, D., Traver, D., Look, A., and Zon, L. (2004) The use of zebrafish to understand immunity. Immunity 20, 367–379.CrossRefPubMedGoogle Scholar
  3. 3.
    Nasevicius, A., and Ekker, S. (2000) Effective targeted “knockdown” in zebrafish. Nat. Genet. 26, 216–220.CrossRefPubMedGoogle Scholar
  4. 4.
    van der Sar, A., Stockhammer, O., van der Laan, C., Spaink, H., Bitter, W., and Meijer, A. (2006) Myd88 innate immune function in a zebrafish embryo infection model. Infect. Immun. 74, 2436–2441.CrossRefPubMedGoogle Scholar
  5. 5.
    Westerfield, M. (1993) The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Brachydanio rerio), The University of Oregon Press, Eugene.Google Scholar
  6. 6.
    van der Sar, A., Musters, R., van Eeden, F., Appelmelk, B., Vandenbroucke-Grauls, C., and Bitter, W. (2003) Zebrafish embryos as a model host for the real time analysis of Salmonella typhimurium infections. Cell. Microbiol. 5, 601–611.CrossRefPubMedGoogle Scholar
  7. 7.
    Herbomel, P., Thisse, B., and Thisse, C. (1999) Ontogeny and behaviour of early macrophages in the zebrafish embryo. Development 126, 3735–45.PubMedGoogle Scholar
  8. 8.
    Herbomel, P., and Levraud, J.P. (2005) Imaging early macrophage differentiation, migration, and behaviors in live zebrafish embryos. Methods Mol. Med. 105, 199–214.PubMedGoogle Scholar
  9. 9.
    Murayama, E., Kissa, K., Zapata, A., Mordelet, E., Briolat, V., Lin, H.F., Handin, R.I., and Herbomel, P. (2000) Tracking hematopridic precursor migration to successive hematopritic organs during zebrafish development. Immunity. 25: 963–975.CrossRefGoogle Scholar
  10. 10.
    Herbomel, P., Thisse, B., and Thisse, C. (2001) Zebrafish early macrophages colonize cephalic mesenchyme and developing brain, retina, and epidermis through a M-CSF receptor-dependent invasive process. Dev. Biol. 238, 274–288.CrossRefPubMedGoogle Scholar
  11. 11.
    Lutfalla, G., and Uzé, G. (2006) Performing quantitative RT-PCR experiments, in DNA Microarrays (Kimmel, A., and Oliver, B., Eds.), Vol. 410, Elsevier Academic Press, San Diego, 383–397.Google Scholar
  12. 12.
    Novak, A., and Ribera, A. (2003) Immunocytochemistry as a tool for zebrafish developmental neurobiology. Methods Cell Sci. 25, 79–83.CrossRefPubMedGoogle Scholar
  13. 13.
    Sheehan, H., and Storey, G. (1947) An improved method of staining leukocyte granules with Sudan black B. J. Pathol. Bacteriol. 59, 336.CrossRefPubMedGoogle Scholar
  14. 14.
    Guryev, V., Koudijs, M., Berezikov, E., Johnson, S., Plasterk, R., van Eeden, F., and Cuppen, E. (2006) Genetic variation in the zebrafish. Genome Res. 16, 497–497.CrossRefGoogle Scholar
  15. 15.
    Lister, J., Robertson, C., Lepage, T., Johnson, S., and Raible, D. (1999) nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate. Development 126, 3757–3767.PubMedGoogle Scholar
  16. 16.
    Lawson, N., and Weinstein, B. (2002) In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev. Biol. 248, 307–318.CrossRefPubMedGoogle Scholar
  17. 17.
    Pauls, S., Geldmacher-Voss, B., and Campos-Ortega, J. (2001) A zebrafish histone variant H2A.F/Z and a transgenic H2A.F/Z:GFP fusion protein for in vivo studies of embryonic development. Dev. Genes Evol. 211, 603–610.CrossRefPubMedGoogle Scholar
  18. 18.
    Kimmel, C., Ballard, W., Kimmel, S., Ullmann, B., and Schilling, T. (1995) Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253–310.PubMedGoogle Scholar
  19. 19.
    Heasman, J. (2002) Morpholino oligos: making sense of antisense? Dev. Biol. 243, 209–214.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2008

Authors and Affiliations

  • Jean-Pierre Levraud
    • 1
  • Emma Colucci-Guyon
    • 1
  • Michael J. Redd
    • 2
  • Georges Lutfalla
    • 3
  • Philippe Herbomel
    • 1
  1. 1.Unité Macrophages et Développement del’ImmunitéInstitut PasteurParisFrance
  2. 2.Huntsman Cancer InstituteSalt Lake CityUT
  3. 3.CNRS/Université Montpellier IIMontpellierFrance

Personalised recommendations